Proteomics

Q Exactive Plus

Mass Range: 50 up to 6,000 m/z (mass over charge)

Resolution: up to 140,000 @ 200 m/z, maximum 280,000

Ion fragmentation: HCD (higher-energy collisional dissociation)

Mass accuracy: < 3 ppm RMS (root mean square) external calibration, < 1 ppm RMS internal calibration

High scan speed (12 Hz, 17,500 resolution)

Multiplexing (up to 10 precursors/scan) and Targeted Proteomics (SIM/PRM)

The Q Exactive Plus is a benchtop size instrument coupled to the Ultimate 3000 RSLC system for LC/MS applications. For electrospray ionization the Proxeon source with a steel emitter is used. Ionized peptides enter the Q Exactive Plus through a heated transfer capillary and an S-lens, focusing the ion stream. The ion stream is then guided to an advanced quadrupole mass filter and selected ions are collected and stored in a C-trap. The C-trap transfers ions either to the Orbitrap mass analyzer or to the HCD cell for ion fragmentation followed by mass analysis in the Orbitrap. The Q Exactive allows selected ion monitoring (SIM) and parallel reaction monitoring (PRM). The instrument is used within the proteomics platform of the TRR130 to provide state-of-the-art LC/MS technology to research projects within the TRR130

Orbitrap Velos Pro Elite

Mass Range: 50 up to 4,000 m/z (mass over charge)

Resolution: up to 200,000 @ 200 m/z, maximum 240,000

Ion fragmentation: HCD (higher-energy collisional dissociation), CID (collisional-induced dissociation), ETD (electron transfer dissociation)

Mass accuracy: < 3 ppm RMS (root mean square) external calibration, < 1 ppm RMS internal calibration

ETD for advanced post-translational modification analysis;

Combination of different fragmentation methods in the same run

Parallel MS and MSn analysis; high-field (HF) Orbitrap

The Orbitrap Velos Pro Elite features two mass analyzers: the linear ion trap and the Orbitrap. The instrument is coupled to the Ultimate 3000 UPLC system and eluting peptides are transferred via a steel emitter to a Proxeon nano-LC electrospray ion source. Charged ions enter the mass spectrometer through a heated transfer capillary and a S-lens for ion focusing. The ions are then transferred through three multipoles to the linear ion trap consisting of a high-pressure cell for peptide accumulation or fragmentation and a low-pressure cell for mass analysis. Through another multipole for transmission the ions reach the C-trap from which they are transferred to either the HCD cell for fragmentation or HF orbitrap mass analyzer for precise mass determination. The installed ETD module allows the fragmentation of peptide ions by electron transfer dissociation. Collisional-induced dissociation (CID) is performed in the linear ion trap and higher-energy collisional dissociation (HCD) in the HCD cell. Due to its two mass analyzers it can simultaneously detect precursor ion as well as product ion masses.

Orbitrap XL

Mass Range: 50 up to 4,000 m/z (mass over charge)

Resolution: up to 60,000 @ 400 m/z, maximum 100,000

Ion fragmentation: CID (collisional-induced dissociation)

Mass accuracy: < 3 ppm RMS (root mean square) external calibration, < 1 ppm RMS internal calibration

The Orbitrap XL is used for routine applications, method optimization and the analysis of samples of low complexity. The instrument features a linear ion trap as well as an orbitrap mass analyzer and is coupled to the Ultmiate3000 RSLC system for LC/MS applications. The eluting peptides are transferred via a fused silica emitter into a Thermo nano-LC source for electrospray ionization. Charged ions enter the mass spectrometer through a heated transfer capillary and are transferred through three multipoles to the linear ion trap for ion storage, selection, and fragmentation by CID. Mass analysis of precursor ions is performed in the orbitrap.

Synapt G1 HDMS Q-TOF system for native MS and ion mobility separation (IMS) of intact proteins and non-covalent protein complexes

Instrument features:

• equipped with nano-electrospray ion source
• adapted to the detection of high masses and intact non-covalent protein complexes
• isolation up to m/z 32,000 for MS/MS
• collision-induced dissociation with argon or xenon gas
• ion mobility separation in a nitrogen-filled drift tube.

The system exploits electrospray to transfer proteins intact and in their folded state as well as non-covalent protein complexes from an aqueous buffer solution into the gas phase without dissociation or fragmentation. This mass spectrometric technology is called “Native MS” and allows for investigating binding affinities and the stoichiometry of protein-protein or protein-ligand complexes in addition to determining the exact molecular masses of the individual components of a given complex. Native MS has recently emerged as an important complementary method of existing structural biology tools. In addition, native MS has been successfully combined with IMS in the gas phase. Here, ions are transferred in packets through a drift tube filled with an inert gas. As the resulting collisions decelerate ions of larger size more than small ones, a drift time spectrum can be recorded. By comparison with the drift times of proteins of known size, collisional cross sections (CCS) are estimated. This allows us to reveal subtle conformational changes induced by ligand binding or mutations or follow the unfolding of proteins upon stepwise collisional activation. Currently, a special focus is on the development of methods for the analysis of membrane protein complexes in the presence of detergent micelles.

Dionex/Thermo Ultimate 3000 systems

We have several differently configured Dionex/Thermo Ultimate 3000 HPLC systems in our lab. The HPLC systems that are used offline (i.e. not directly coupled to a mass spectrometer) have milliliter to microliter flow rates. In combination with different analytical columns they are used for reversed-phase (RP) or size exclusion chromatography of intact proteins or peptides after enzymatic digestion. The LC systems also contain a sample tray module that allows the collection of the chromatographic fractions. The online HPLC systems provide nanoliter flowrates that are required for stable electrospray ionization. They are equipped with a double valve system that offers the parallel loading of the sample on one of the two pre-columns while the other pre-column is washed and conditioned. Peptide separation is achieved by a RP analytical column and the peptide signal can be directly monitored with an UV detector prior to electrospray ionization and subsequent mass analysis.